The long-term goal of this project is to explore mechanisms linked to lipid-induced insulin resistance. A common theme emerging is that mitochondrial function is impaired in these conditions. In recent studies we discovered chronic high fat feeding resulted in diminished mitochondrial respiratory capacity at a time when incomplete [unreadable]-oxidation was accelerated, thus implying a disconnect between [unreadable]-oxidation and tricarboxylic acid cycle activity. We hypothesize that the mitochondrial matrix enzyme carnitine acetyltransferase (CrAT), through its action allowing the export of excess incomplete [unreadable]-oxidative intermediates from the mitochondria, plays a critical role in maintaining skeletal muscle mitochondrial function under periods of excess lipid burden. Using adenoviral-mediated silencing of CrAT in skeletal muscle cell culture models (L6 and C2C12 cell lines), as well as muscle specific targeted silencing of CrAT (adenoviral injection or electroporation) in mice, we will explore the role of CrAT in mitochondrial substrate handling. Classic functional assays and mass spectrometry-based metabolic profiling will be employed to determine whether siCrAT treatment increases susceptibility to lipid-induced mitochondrial dysfunction and subsequently predisposes skeletal muscle toward the development of insulin resistance. Provided a phenotype is observed we will explore whether CrAT modulates mitochondrial protein acetylation &/or oxidative stress to assess potential mechanisms behind the role of this enzyme in regulating mitochondrial function in response to a high lipid environment. Results from these experiments will provide valuable insights into the role of CrAT in lipid-induced mitochondrial dysfunction and insulin resistance. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: The incidence of obesity, insulin resistance, and diabetes are increasing at alarming rates and are closely associated with dysregulation of lipid metabolism and mitochondrial function. CrAT, an enzyme localized within the mitochondrial matrix, converts short chain acyl-CoAs to short chain acylcarnitines which can then be exported from the mitochondria, thereby providing a mechanism to maintain mitochondrial function under conditions of lipid excess. The proposed experiments will greatly enhance our understanding of the role of CrAT in the regulation of mitochondrial substrate handling, as well as establishing a potential role of this enzyme in the alleviation of obesity, insulin resistance, and diabetes. [unreadable] [unreadable] [unreadable]